Antisense oligonucleotides (hereinafter, "ASO"s) are short, usually synthetic, nucleic acids designed to bind to mRNA or other nucleic acids comprising specific sequences, taking advantage of Watson-Crick-type base pairing. Prior art ASO therapeutic strategies are designed to suppress the expression of specific genes involved in cancer, inflammatory diseases, and viral infections (Crooke et al., 1996, Annu. Rev. Pharmacol. Toxicol. 36:107-129). More than ten ASOs are currently undergoing human clinical trials for the treatment of various diseases (Matteucci et al., 1996, Nature 384(Supp.):20-22; Agrawal, 1996, Trends Biotechnol. 14: 376-387).
Antisense therapy comprising binding of an ASO to mRNA in a cell affected by a disease or disorder has, to date, been a therapeutic strategy wherein it has been difficult to identify efficacious target sites for a given RNA sequence (Gewirtz et al., 1996, Proc. Natl. Acad. Sci. U.S.A. 93:3161-3163). A significant shortcoming of prior art antisense strategies is the inability to accurately predict which ASOs will prove efficacious among a population of potentially efficacious ASOs (Laptev et al., 1994, Biochemistry 33:11033-11039). Because prior art attempts to predict the therapeutic efficacy of ASOs have been largely unsuccessful, selection of ASO sequences for antisense therapy has, prior to the present disclosure, been performed by empirically screening large numbers of potential antisense agents (Bennett et al., 1994, J. Immunol. 152:3530-3540). Using trial-and-error ASO selection strategies of the prior art, a large number of ASOs must be tested in order to discover a few sequences which exhibit significant efficacy as therapeutic ASOs. Prior art strategies require the screening of large numbers of ASOs because any portion of an mRNA molecule can be used to design a complementary ASO.
For example, an mRNA molecule which consists of 2000 nucleotide residues affords 1980 potential target sites for an ASO comprising twenty-one nucleotides which is complementary to twenty-one sequential nucleotide residues of the mRNA molecule. The trial-and-error methods of the prior art ASO selection process therefore recommend the manufacture and assay of at least 30-40 potential ASOs in order to identify likely no more than a few efficacious ASOs. Clearly, a method of designing ASOs which reduces or avoids dependence on trial-and-error selection methods would be of great value by reducing the duration and expense of ASO development efforts.
Investigations have been made by others to determine the effect upon efficacy of designing ASOs complementary to various regions of mRNA molecules. In general, these investigations have concentrated on complementation of an ASO to a discrete region within MRNA molecules. For example, various investigators have determined that efficacious ASOs may be constructed which are complementary:
a) to regions encompassing the 5'-cap site of an mRNA molecule (Ojala et al., 1997, Antisense Nucl. Drug Dev. 7:31-38), PA0 b) to regions encompassing the transcription start site (Monia et al., 1992, J. Biol. Chem. 267:19954-19962), PA0 c) to regions encompassing the translation initiation codon (Dean et al., 1994, Proc. Natl. Acad. Sci. U.S.A. 91:11762-11766), PA0 d) to regions encompassing the translation stop codon (Wang et al., 1995, Proc. Natl. Acad. Sci. USA 92:3318-3322), PA0 e) to regions encompassing sites at which mRNA molecules are spliced (Agrawal et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 86:7790-7794; Colige et al., 1993, Biochem. 32:7-11), PA0 f) to regions encompassing the 5'-untranslated region of mRNA molecules (Duff et al., 1995, J. Biol. Chem. 270:7161-7166; Yamagami et al., 1996, Blood 87:2878-2884), PA0 g) to regions encompassing the 3'-untranslated region of mRNA molecules (Bennett et al., 1994, J. Immunol. 152:3530-3540; Dean et al., 1994, J. Biol. Chem. 269:16146-16424), and PA0 h) to regions encompassing the coding region (Laptev et al., 1994, Biochem. 33:11033-11039; Yamagami et al., 1996, Blood 87:2878-2884).
Because efficacious ASOs may, as demonstrated by these investigators, be complementary to any region of an mRNA molecule, the ASO designer is not provided any meaningful guidance by these studies.
Several strategies have been proposed to facilitate and simplify the selection process for efficacious ASOs. One strategy relies upon predictions of the binding energy between an ASO and a complementary sequence in an mRNA molecule. Chiang et al. (1991, J. Biol. Chem. 266:18162-18171) designed ten ASOs complementary to mRNA encoding human ICAM-1 protein with the aid of the computer program, OLIGO. These ten oligonucleotides were designed to maximize the melting temperature (T.sub.m) of the oligonucleotide-mRNA complex. However, these investigators discovered that the efficacy of the ASOs as inhibitors of ICAM-1 expression did not correlate directly with either the Tm of the oligonucleotide-mRNA complex or the .DELTA.G.degree..sub.37 (change in free energy upon association/dissociation of the oligonucleotide and the mRNA complex, as assessed at 37.degree. C.). The most potent oligonucleotide (ISIS 1939) identified by these investigators exhibited a T.sub.m value that was lower than those corresponding to the majority of the other oligonucleotides which were tested. Thus, maximization of binding energy between an ASO and a complementary mRNA is not sufficient to ensure therapeutic efficacy of the oligonucleotide.
Stull et al. (1992, Nucl. Acids Res. 20:3501-3508) investigated a systematic approach for predicting appropriate sequences within an mRNA molecule against which complementary ASOs could be constructed, by calculating three thermodynamic indices: (i) a secondary structure score (Sscore), (ii) a duplex score (Dscore); and (iii) a competition score (Cscore), which is the difference between the Dscore and the Sscore. The Sscore estimates the strength of local mRNA secondary structures at the mRNA binding site for the ASO. The Dscore estimates AGformation, the change in Gibbs free energy upon formation of the duplex, of the oligonucleotide-mRNA target sequence duplex. These three indices were compared to the efficacy of ASOs for inhibiting protein expression. It was found that the Dscore was the most consistent predictor of ASO efficacy in four of the five studies (the correlation factor r.sup.2 ranged from 0.44 to 0.99 in these four studies). The results of the fifth study could not be predicted by any thermodynamic or physical index.
A second strategy for selecting efficacious ASOs is based upon predicting the secondary structure of mRNA. Wickstrom and colleagues (1991, In Prospects for antisense nucleic acid therapy of cancer and AIDS, Wickstrom, ed., Wiley-Liss, Inc., New York, 7-24) attempted to correlate the efficacy of potential ASOs with the secondary structure of the complementary region of the mRNA. It was hypothesized that ASOs would be the most efficacious when they were designed to be complementary to the target sequences within the mRNA molecule which were the least involved in the secondary and tertiary structure of the mRNA molecule. These investigators designed fourteen ASOs which were complementary to the predicted stems, loops, and bulges of human C-myc p65 mRNA. ASOs were designed which were complementary to regions of the mRNA molecule between the 5'-cap site and the translation initiation codon AUG, and included oligonucleotides which were complementary to sequences located within a predicted hairpin sequence which was located immediately 3' to the AUG initiation codon. These investigators discovered that two fragments, one comprising the 5'-cap sequence and the other comprising a sequence located slightly 3' relative to the cap sequence, were better target sequences for ASOs than the sequence spanning the AUG initiation codon, even though the sequence spanning the AUG initiation codon was located at an even weaker bulge and stem area.
Lima et al. (1992, Biochem. 31:12055-12061) designed six ASOs, each of which was complementary to a portion of a 47-nucleotide region that was able to achieve a stable hairpin conformation within an activated Ha-ras gene transcript. These investigators discovered that two of the oligonucleotides which were complementary to the loop portion of the hairpin structure had nearly equal binding affinity for the transcript. In contrast, they observed that oligonucleotides which were complementary to the double-stranded stem portion of the hairpin structure were less tightly bound, having affinity constants that were smaller by a factor of between 10.sup.5 and 10.sup.6. These results suggest that mRNA sequences which lie within regions of secondary structure may be undesirable target sequences for designing complementary ASO.
Thierry et al. (1993, Biochem. Biophys. Res. Commun. 190:952-960) compared the efficacy of ASOs which were complementary to either the 5'-end of the coding region of or to a single-stranded loop in the mRNA encoded by the multidrug resistance gene mdr1. The results obtained by these investigators indicate that the oligonucleotides targeted to the single-stranded loop were more efficacious and specific than the oligonucleotides targeted to the 5'-end coding region. However, Laptev et al. (1994, Biochem. 33:11033-11039) obtained results which were not consistent with that suggestion. Laptev et al. concluded that the most efficacious ASOs were those which were complementary to mRNA sequences that were predicted to form clustered double-stranded secondary structures.
Still other investigators presented evidence that the most efficacious ASOs (ISIS 1939 and ISIS 2302) were those which were complementary to regions of human ICAM-1 mRNA, which regions were predicted by computer modeling to form stable stem-loop structures (Chiang et al., 1991, J. Biol. Chem. 266:18162-18171; Bennett et al., 1994, J. Immunol. 152:3530-3540). Oligonucleotides which were complementary to mRNA sequences upstream or downstream from these putative stem-loop structures had significantly less inhibitory activity (Bennett et al., 1994 Adv. Pharmacol. 28:1).
Fenster et al. (1994, Biochemistry 33, 8391-8398) observed that inhibition of gene expression by an ASO was highly dependent upon the position of the mRNA sequence to which the oligonucleotide was complementary. These investigators discovered that the most potent ASOs to effect inhibition of the Rev-response element of the human immunodeficiency virus (HIV) were complementary to mRNA target sites corresponding to the stem-loop V region of the HIV mRNA. This region of the HIV mRNA is known to be important for full and efficient Rev-response element function. ASOs targeted to other, noncritical Rev-response element stem-loops (e.g. SLI and SLIII) were determined by these investigators to be either non-efficacious or 30-fold less efficacious than stem-loop V oligonucleotides for inhibiting Rev-response element function.
Hence, it is clear that the bulk of ASO selection strategies reported in the prior art have been directed to designing ASOs which are complementary to discrete regions within mRNA molecules, rather than to particular sequences within mRNA.
Recently, Ho et al. (1996, Nucl. Acids Res. 24:1901-1907; 1998, Nature Biotechnol. 16:59-63) developed a novel approach to rationally select ASOs. These investigators contacted an mRNA molecule encoding human multidrug resistance-1 protein and an mRNA molecule encoding angiotensin type I receptor protein with a library of chimeric oligonucleotides. Hybridized mRNA was subsequently treated with RNase H, an enzyme which catalyzes the hydrolytic cleavage of only the RNA strand of an RNA-DNA duplex. The RNA fragments which were generated were sequenced to identify regions on the mRNA sequence which were involved in RNA-DNA duplex formation. Using the sequence information, these investigators constructed ASOs which were complementary to these regions and found those particular ASOs to be significantly more efficacious than randomly-selected oligonucleotides for inhibiting human multidrug resistance-1 protein or angiotensin type I receptor protein expression. These results demonstrate that it is feasible to construct improved ASOs by incorporating therein sequences which are complementary to particular nucleotide sequences found in mRNA molecules.
Skilled workers in the art have concluded the therapeutic efficacy of an ASO which is complementary to a particular target sequence within an mRNA molecule has not heretofore been accurately predictable (Gewirtz et al., 1996, Proc. Natl. Acad. Sci. U.S.A. 93:3161-3163). Because efficacious ASOs have been made which are complementary to most or all regions of mRNA molecules, the ASO designer cannot be meaningfully guided by selection of any particular mRNA region. Methods for predicting the efficacy of ASOs by maximization of Tm or AGformation have not consistently yielded correct predictions, and thus are similarly of limited use to the ASO designer. Analyses of the secondary structure of an mRNA do not clearly identify potential ASO-binding sites. Library-based RNaseH degradation studies are laborious and complex. Other skilled workers in the art have recognized that a long-felt, but unmet, need exists for methods of selecting the most potent target sequences within a given mRNA sequence (Szoka, 1997, Nature Biotechnol. 15:509).
In April of 1998, results of a consensus reached at U.S. National Institutes of Health conference on this subject were reported. These results included the conclusions that "[i]t appears that the only way to generate an active oligomer is by brute force" and that "[o]ptimally it is best to screen 30-40 oligos to obtain one species that is maximally active, but this may be impossible because of time and cost considerations" (Stein, 1998, Antisense and Nucleic Acid Drug Development 8: 129-132).
Taken together, the results of these prior art methods for designing an ASO sequence offer little guidance to the ASO designer. The present invention overcomes the shortcomings of prior art ASO design methods by providing a method for designing efficacious ASOs.